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- EMDB-8855: Cryo-EM structure of P. aeruginosa flagellar filaments A443V -

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Basic information

Entry
Database: EMDB / ID: EMD-8855
TitleCryo-EM structure of P. aeruginosa flagellar filaments A443V
Map dataCryo-EM structure of P. aeruginosa flagellar filaments A443V
Sample
  • Complex: Pseudomonas aeruginosa flagella filament
    • Protein or peptide: B-type flagellin
Keywordsbacteria flagella / helical polymers / cryo-EM / PROTEIN FIBRIL
Function / homology
Function and homology information


bacterial-type flagellum / bacterial-type flagellum-dependent cell motility / structural molecule activity / extracellular region
Similarity search - Function
Flagellin hook, IN motif / Flagellin hook IN motif / Flagellin, C-terminal domain, subdomain 2 / Flagellin, C-terminal domain / Bacterial flagellin C-terminal helical region / Flagellin / Flagellin, N-terminal domain / Bacterial flagellin N-terminal helical region
Similarity search - Domain/homology
Biological speciesPseudomonas aeruginosa PAO1 (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 4.2 Å
AuthorsWang F / Postel S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM122510 United States
CitationJournal: Nat Commun / Year: 2017
Title: A structural model of flagellar filament switching across multiple bacterial species.
Authors: Fengbin Wang / Andrew M Burrage / Sandra Postel / Reece E Clark / Albina Orlova / Eric J Sundberg / Daniel B Kearns / Edward H Egelman /
Abstract: The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we ...The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we present near-atomic resolution cryo-EM structures for flagellar filaments from both Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa. Seven mutant flagellar filaments in B. subtilis and two in P. aeruginosa capture two different states of the filament. These reliable atomic models of both states reveal conserved molecular interactions in the interior of the filament among B. subtilis, P. aeruginosa and Salmonella enterica. Using the detailed information about the molecular interactions in two filament states, we successfully predict point mutations that shift the equilibrium between those two states. Further, we observe the dimerization of P. aeruginosa outer domains without any perturbation of the conserved interior of the filament. Our results give new insights into how the flagellin sequence has been "tuned" over evolution.Bacterial flagellar filaments are composed almost entirely of a single protein-flagellin-which can switch between different supercoiled states in a highly cooperative manner. Here the authors present near-atomic resolution cryo-EM structures of nine flagellar filaments, and begin to shed light on the molecular basis of filament switching.
History
DepositionJul 24, 2017-
Header (metadata) releaseAug 30, 2017-
Map releaseOct 25, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.76
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.76
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5wk5
  • Surface level: 0.76
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5wk5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8855.png

Downloads & links

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Map

FileDownload / File: emd_8855.map.gz / Format: CCP4 / Size: 184.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of P. aeruginosa flagellar filaments A443V
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 1000 pix.
= 1050. Å		1.05 Å/pix.
x 220 pix.
= 231. Å		1.05 Å/pix.
x 220 pix.
= 231. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.76 / Movie #1: 0.76
Minimum - Maximum-1.5654951 - 2.5079873
Average (Standard dev.)0.070206635 (±0.36481166)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-110-110-500
Dimensions2202201000
Spacing2202201000
CellA: 230.99998 Å / B: 230.99998 Å / C: 1050.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z2202201000
origin x/y/z0.0000.0000.000
length x/y/z231.000231.0001050.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-110-110-500
NC/NR/NS2202201000
D min/max/mean-1.5652.5080.070

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Supplemental data

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Sample components

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Entire : Pseudomonas aeruginosa flagella filament

EntireName: Pseudomonas aeruginosa flagella filament
Components
  • Complex: Pseudomonas aeruginosa flagella filament
    • Protein or peptide: B-type flagellin

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Supramolecule #1: Pseudomonas aeruginosa flagella filament

SupramoleculeName: Pseudomonas aeruginosa flagella filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Macromolecule #1: B-type flagellin

MacromoleculeName: B-type flagellin / type: protein_or_peptide / ID: 1 / Number of copies: 41 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 49.302906 KDa
Recombinant expressionOrganism: Pseudomonas aeruginosa PAO1 (bacteria)
SequenceString: MALTVNTNIA SLNTQRNLNA SSNDLNTSLQ RLTTGYRINS AKDDAAGLQI SNRLSNQISG LNVATRNAND GISLAQTAEG ALQQSTNIL QRIRDLALQS ANGSNSDADR AALQKEVAAQ QAELTRISDT TTFGGRKLLD GSFGTTSFQV GSNAYETIDI S LQNASASA ...String:
MALTVNTNIA SLNTQRNLNA SSNDLNTSLQ RLTTGYRINS AKDDAAGLQI SNRLSNQISG LNVATRNAND GISLAQTAEG ALQQSTNIL QRIRDLALQS ANGSNSDADR AALQKEVAAQ QAELTRISDT TTFGGRKLLD GSFGTTSFQV GSNAYETIDI S LQNASASA IGSYQVGSNG AGTVASVAGT ATASGIASGT VNLVGGGQVK NIAIAAGDSA KAIAEKMDGA IPNLSARART VF TADVSGV TGGSLNFDVT VGSNTVSLAG VTSTQDLADQ LNSNSSKLGI TASINDKGVL TITSATGENV KFGAQTGTAT AGQ VAVKVQ GSDGKFEAAA KNVVAAGTAA TTTIVTGYVQ LNSPTAYSVS GTGTQASQVF GNASAAQKSS VASVDISTAD GAQN AIAVV DNALAAIDAQ RADLGAVQNR FKNTIDNLTN ISENATNVRS RIKDTDFAAE TAALSKNQVL QQAGTAILAQ ANQLP QAVL SLLR

UniProtKB: B-type flagellin

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4 / Details: PBS
StainingType: NEGATIVE / Material: negative stain
GridPretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 2.0 sec. / Average electron dose: 20.0 e/Å2
Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the ...Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the estimation of the CTF as well as for boxing filaments. Only the first two parts were used for the reconstruction (~5 electrons per Angstrom^2).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 4.61 Å
Applied symmetry - Helical parameters - Δ&Phi: 65.75 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: OTHER / Software - Name: SPIDER / Details: model-map FSC 0.38 cut-off / Number images used: 102119
Startup modelType of model: OTHER / Details: featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-5wk5:
Cryo-EM structure of P. aeruginosa flagellar filaments A443V

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